Commonly used protein expression systems include those derived from bacteria, yeast, baculovirus/insect cells, and mammalian cells. Bacterial expression systems, such as Escherichia coli, have the advantage of fast growth kinetics, easily achieving high cell density cultures, and fast and easy transformation with exogenous DNA. However, current bacterial expression systems often result in low protein yields or unstable proteins.
Energy-dependent degradation in Escherichia coli is carried out by a number of ATP-dependent proteases, including Lon, FtsH, and different Clp proteases. Gottesman S., (1996) Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30:465-506. The Clp proteases are composed of two different multimeric components. The smaller subunit is a peptidase, either ClpP or ClpQ (HslV). Maurizi et al., (1990) Sequence and structure of ClpP, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J. Biol. Chem. 265:12536-12545. Missiakas et al., Identification and characterization of HslV HslU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J. 15:6899-6909. The larger subunit is an ATPase, ClpA, ClpX, or ClpY (also called HslU). Katayama-Fujimura et all, (1987) A multiple-component ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262:4477-4485. Gottesman et al., (1993) ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. J. Biol. Chem. 268:22618-22626. Rohrwild et al., (1996) HslV-HslU: a novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc. Natl. Acad. Sci. USA 93:5808-5813. Wu, et al., Redundant In Vivo Proteolytic Activities of Escherichia coli Lon and the ClpYQ (HslUV) Protease, J. Bacteriol. June 1999 vol. 181 no. 12 3681-3687.
Either ClpA or ClpX can associate with ClpP to form an ATP-dependent protease (ClpAP or ClpXP). ClpY associates with ClpQ to form an active, energy-dependent protease. While ClpY and ClpX show significant sequence similarity to each other and to ClpA, ClpP and ClpQ(HslV) are not related at the sequence level. ClpP is a serine protease, found in many prokaryotes and in the organelles of many eukaryotes; ClpQ has a catalytic amino-terminal threonine residue and shows sequence similarity to the eukaryotic proteasome P subunit. Seemuller et al., (1995) Proteasome from Thermoplasma acidophilum: a threonine protease. Science, 268:579-582.
Like other parental E. coli B strains, BL21 cells are deficient in the Lon protease, which degrades many foreign proteins. Gottesman S. (1996) Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30 465-506. Another gene missing from the genome of the ancestors of BL21 is the outer membrane protease OmpT, whose function is to degrade extracellular proteins. In the BL21(DE3) strain, the λDE3 prophage was inserted in the chromosome of BL21 and contains the T7 RNAP gene under the lacUV5 promoter. The BL21(DE3) and its derivatives, as well as the K-12 lineage are widely used for protein expression. Rosano et al., Recombinant protein expression in Escherichia coli: advances and challenges, Front Microbiol. 2014, 5:172.
The present disclosure relates to bacteria that have high levels of protein expression due to disruption of at least one protease. The present disclosure also provides for a method to improve the yield of expressed recombinant proteins, including unstable proteins.